The present invention relates to compounds and methods for inhibiting cell death, such as neuronal or myocardial cell death. The compounds and pharmaceutical compositions thereof are particularly effective in inhibiting apoptotic cell death, and thus may be used to protect cells from cell death associated with ischemia, trauma, neurodegeneration, and inflammation.
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Apoptosis has been associated with ischemic injury, such as typically occurs in cases of stroke, myocardial infarction, and reperfusion injury (Walton et al., 1997; MacManus et al., 1993). Apoptosis is also associated with immunoreactive and immunodegenerative states and a variety of neurodegenerative disorders. Recent studies on the mechanism of retinal ganglion cell death in experimental glaucoma also indicate that the cells die by apoptosis (Nickells, 1996; Garcia-Valenzuela et al., 1995; Laquis et al., 1998).
Apoptosis is a programmed cell death, occurring in normally functioning human and animal cells when age or state of cell health and condition dictates. It is an active process requiring metabolic activity by the dying cell, and is often characterized by cleavage of the DNA into fragments that give a so called laddering pattern on gels. Cells that die by apoptosis do not usually elicit the inflammatory responses that are associated with necrosis, a passive process in which collapse of internal homeostasis leads to cellular dissolution.
Apoptosis can have particularly devastating consequences when it occurs pathologically in cells that do not normally regenerate, such as neurons. Because such cells are not replaced when they die, their loss can lead to debilitating and sometimes fatal dysfunction of the affected organ.
Various drug strategies have been proposed for treatment of stroke and other neuronal conditions related to ischemia. To date, however, these drugs have been either relatively ineffective or effective only at dosage levels where undesired side effects are observed. For example, anti-coagulants, such as heparin, antivasoconstriction agents, such as flunarazine, excitatory neurotransmitter antagonists, such as MK-801 and AP7, and anti-edemic compounds have shown mixed results, with no clear benefits to outweigh a variety of side effects, including neurotoxicity or increased susceptibility to infection. Verapamil and related compounds, which prevent calcium entry into smooth and striated muscles, appear to be effective only at high drug concentrations, where serious cardiotoxicity effects may ensue. Increased cerebral edema has been observed as a side effect in treatment with dihydropyridines, such as nimodipine. Benzothiazepines, as exemplified by diltiazem, have shown moderate protective effects, but these drugs also appear to cause undesired side effects, such as hypotension, which may be inimical to treatment.
In one aspect, the invention provides a pharmaceutical composition, useful for inhibiting cell death, which comprises an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 
In formula I, X, Xxe2x80x2, Z and Zxe2x80x2 are independently selected from the group consisting of hydrogen, alkyl, alkoxy, cyano, carboxylic acid or ester, sulfonic acid or ester, amino, alkylamino, nitro, and halogen. The linker L is NR1, carbonyl, CR2R3, or a direct bond, where R1 and R2 are independently selected from hydrogen, alkyl, aryl, and aralkyl, and R3 is selected from hydrogen, lower alkyl, amino, lower alkylamino, nitro, halogen, and lower alkyl sulfonate. The moiety AB represents a three-atom linkage effective to form an imidazole, pyrrole, oxazole or thiazole ring fused to the adjacent six-membered ring, where one of A and B is nitrogen or carbon and the other is selected from NR1, O, or S, wherein at least one of A and B is nitrogen, and where AB groups on opposing sides of the linker L may be the same or different. The groups Y and Yxe2x80x2 are independently selected from carbon and nitrogen.
In selected embodiments, the linker L is CH2, CHCH3, or carbonyl, and is preferably CH2. In further embodiments, in which AB represents a three-atom linkage effective to form an imidazole ring fused to the adjacent six-membered ring, NR1 is preferably NH, NCH3, or NCH2C6H5 (N-benzyl). Y and Yxe2x80x2 are preferably carbon.
In further embodiments, X, Xxe2x80x2, Z and Zxe2x80x2 are independently selected from hydrogen, alkyl, carboxylic acid or ester, amino, nitro, chloro, and fluoro. Preferably, at least one of X and Xxe2x80x2 is amino or nitro, and Z and Zxe2x80x2 are independently selected from hydrogen, carboxylic acid, chloro, and fluoro. Not included are compositions in which, in Formula I, L is CH2, Y is carbon, AB represents a three-atom linkage effective to form an imidazole or pyrrole ring, X, Xxe2x80x2 and R1 are hydrogen, and Z and Zxe2x80x2 are each selected from hydrogen, nitro, amino, or halogen. However, methods of administering these compositions to inhibit cell death, as described below, are included in the invention.
Alternatively, the pharmaceutical compositions of the invention may comprise an effective amount of a compound of formula II, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 
In formula II, L is NR1, carbonyl, CR2R3, or a direct bond, where R1 and R2 are independently selected from hydrogen, alkyl, aryl, and aralkyl, and R3 is selected from hydrogen, lower alkyl, amino, lower alkylamino, nitro, halogen, and lower alkyl sulfonate. R4 is selected from hydrogen, alkyl, aryl, and aralkyl; and R5 is selected from an electron pair, hydrogen, alkyl, aryl, and aralkyl. It is understood that when R5 is not an electron pair, the compound has a positive charge (e.g. compound SNX-980). L is preferably CR2R3, where R2 and R3 are selected independently from hydrogen and lower alkyl.
As in formula I, AB represents a three-atom linkage effective to form an imidazole, pyrrole, oxazole or thiazole ring fused to the adjacent six-membered ring, where one of A and B is nitrogen or carbon and the other is selected from NR1, O, or S, wherein at least one of A and B is nitrogen; and Y is carbon or nitrogen.
The group W represents a two- to four-carbon alkyl chain linking the two depicted nitrogen atoms to form a five- to seven-membered heterocyclic ring. Each carbon atom of the alkyl chain is unsubstituted or substituted with one or two lower alkyl groups or a hydroxyl group. Preferably, each carbon atom of the alkyl chain is unsubstituted or methyl substituted.
Z represents one or more substituents on the aryl ring containing Y, independently selected from the group consisting of hydrogen, alkyl, alkoxy, cyano, carboxylic acid or ester, sulfonic acid or ester, amino, nitro, and halogen. Preferably, Z is selected from hydrogen, methyl, amino, nitro, chloro, and fluoro.
In selected embodiments, AB represents a three-atom linkage effective to form an imidazole ring fused to the adjacent six-membered ring, and Y and Yxe2x80x2 are carbon. In further embodiments, R4 is selected from hydrogen, lower alkyl, and benzyl, and R5 is an electron pair.
Alternatively, the pharmaceutical compositions may comprise an effective amount of a compound of formula III, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 
In formula III, L is NR1, carbonyl, CR2R3, or a direct bond, where R1 and R2 are independently selected from hydrogen, alkyl, aryl, and aralkyl, and R3 is selected from hydrogen, lower alkyl, amino, lower alkylamino, nitro, halogen, and lower alkyl sulfonate; each R4 is independently selected from hydrogen, alkyl, aryl, and aralkyl; and M is xe2x80x94CR6R7xe2x80x94CR8R9xe2x80x94 or xe2x80x94CR6xe2x95x90CR8xe2x80x94, where R6xe2x80x94R9 are independently selected from hydrogen and lower alkyl.
In selected embodiments, L is CH2, CHNH2, CHNO2, carbonyl, or a direct bond. In further embodiments, R4 is hydrogen or lower alkyl. In one embodiment, M is xe2x80x94CR6xe2x95x90CR8xe2x80x94, and R6 and R8 are independently hydrogen or methyl.
In a further embodiment, the pharmaceutical compositions comprise an effective amount of a compound of formula IV, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 
In formula IV, as above, AB represents a three-atom linkage effective to form an imidazole, pyrrole, oxazole or thiazole ring fused to the adjacent six-membered ring, where one of A and B is nitrogen or carbon and the other is selected from NR1, O, or S, wherein at least one of A and B is nitrogen, and Y is carbon or nitrogen. Z represents one or more substituents on the aryl ring containing Y, independently selected from the group consisting of hydrogen, alkyl, alkoxy, cyano, carboxylic acid or ester, sulfonic acid or ester, amino, nitro, and halogen. Q is selected from nitro and 2-pyridyl. Preferably, Y is carbon, and AB represents a three-atom linkage effective to form an imidazole ring fused to the adjacent six-membered ring. In selected embodiments, Z is hydrogen.
In another aspect, the invention provides a method of inhibiting cell death. In accordance with the method, an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier, is administered to a subject in need of such treatment. 
In formula I, as described above, X, Xxe2x80x2, Z and Zxe2x80x2 are independently selected from the group consisting of hydrogen, alkyl, alkoxy, cyano, carboxylic acid or ester, sulfonic acid or ester, amino, alkylamino, nitro, and halogen; L is NR1, carbonyl, CR2R3, or a direct bond, where R1 and R2 are independently selected from hydrogen, alkyl, aryl, and aralkyl, and R3 is selected from hydrogen, lower alkyl, amino, lower alkylamino, nitro, halogen, and lower alkyl sulfonate; AB represents a three-atom linkage effective to form an imidazole, pyrrole, oxazole or thiazole ring fused to the adjacent six-membered ring, where one of A and B is nitrogen or carbon and the other is selected from NR1, O, or S, wherein at least one of A and B is nitrogen, and where AB groups on opposing sides of the linker L may be the same or different; and Y and Yxe2x80x2 are independently selected from carbon and nitrogen.
In selected embodiments, the linker L is CH2, CHCH3, or carbonyl, and is preferably CH2. In further embodiments, in which AB represents a three-atom linkage effective to form an imidazole ring fused to the adjacent six-membered ring, NR1 is preferably NH, NCH3, or NCH2C6H5 (N-benzyl). Y and Yxe2x80x2 are preferably carbon.
In further embodiments, X, Xxe2x80x2, Z and Zxe2x80x2 are independently selected from hydrogen, alkyl, carboxylic acid or ester, amino, nitro, chloro, and fluoro. Preferably, at least one of X and Xxe2x80x2 is amino or nitro, and Z and Zxe2x80x2 are independently selected from hydrogen, carboxylic acid, chloro, and fluoro.
Alternatively, the method of the invention comprises administering an effective amount of a compound of formula II, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 
In formula II, as described above, L is NR1, carbonyl, CR2R3, or a direct bond, where R1 and R2 are independently selected from hydrogen, alkyl, aryl, and aralkyl, and R3 is selected from hydrogen, lower alkyl, amino, lower alkylamino, nitro, halogen, and lower alkyl sulfonate. R4 is selected from hydrogen, alkyl, aryl, and aralkyl; and R5 is selected from an electron pair, hydrogen, alkyl, aryl, and aralkyl. It is understood that when R5 is not an electron pair, the compound has a positive charge (e.g. compound SNX-980). L is preferably CR2R3, where R2 and R3 are selected independently from hydrogen and lower alkyl.
AB. represents a three-atom linkage effective to form an imidazole, pyrrole, oxazole or thiazole ring fused to the adjacent six-membered ring, where one of A and B is nitrogen or carbon and the other is selected from NR1, O, or S, wherein at least one of A and B is nitrogen; Y is carbon or nitrogen.
W represents a two- to four-carbon alkyl chain linking the two depicted nitrogen atoms to form a five- to seven-membered heterocyclic ring. Each carbon atom of the alkyl chain is unsubstituted or substituted with one or two lower alkyl groups or a hydroxyl group. Preferably, each carbon atom of the alkyl chain is unsubstituted or methyl substituted.
Z represents one or more substituents on the aryl ring containing Y, independently selected from the group consisting of hydrogen, alkyl, alkoxy, cyano, carboxylic acid or ester, sulfonic acid or ester, amino, nitro, and halogen. Selected embodiments of the compounds of formula II which may be employed in the method are described above.
Alternatively, the method of the invention comprises administering an effective amount of a compound of formula III, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 
In formula III, L is NR1, carbonyl, CR2R3, or a direct bond, where R1 and R2 are independently selected from hydrogen, alkyl, aryl, and aralkyl, and R3 is selected from hydrogen, lower alkyl, amino, lower alkylamino, nitro, halogen, and lower alkyl sulfonate; each R4 is independently selected from hydrogen, alkyl, aryl, and aralkyl; and M is xe2x80x94CR6R7xe2x80x94CR8R9xe2x80x94 or xe2x80x94CR6xe2x95x90CR8xe2x80x94, where R6xe2x80x94R9 are independently selected from hydrogen and alkyl. Selected embodiments of the compounds of formula III which may be employed in the method are described above.
In a further embodiments, the method of the invention comprises administering an effective amount of a compound of formula IV, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier. 
In formula IV, AB represents a three-atom linkage effective to form an imidazole, pyrrole, oxazole or thiazole ring fused to the adjacent six-membered ring, where one of A and B is nitrogen or carbon and the other is selected from NR1, O, or S, wherein at least one of A and B is nitrogen; Y is carbon or nitrogen; Z represents one or more substituents on the aryl ring containing Y, independently selected from the group consisting of hydrogen, alkyl, alkoxy, cyano, carboxylic acid or ester, sulfonic acid or ester, amino, nitro, and halogen; and Q is selected from the group consisting of hydrogen, nitro, cyano, and 2-pyridyl. Selected embodiments of the compounds of formula IV which may be employed in the method are described above.
In one embodiment of the present method, the cell death being treated or prevented is apoptotic neuronal cell death, such as that associated with stroke, ischemia, neurodegeneration, trauma, an autoimmune response, or inflammation. In another embodiment, the cell death is associated with myocardial damage, such as myocardial infarction and the resulting ischemia, hypoxia and subsequent reperfusion in the affected area, or myocardial damage resulting from therapeutic intervention, e.g. coronary arterial bypass graft (CABG) or percutaneous transluminal coronary angioplasty (PTCA; xe2x80x9cballoonxe2x80x9d angioplasty).
In a further embodiment of the method, the compounds of formulas I-IV are administered in combination with an anti-hypertensive agent, an antibiotic, an inmmunomodulator, or an anti-inflammatory agent.
Also included within the invention are certain methylenebis(benzimidazole) compounds of formula I above, where L is CH2, Y and Yxe2x80x2 are carbon, and AB represents a three-atom linkage effective to form an pyrrole ring fused to the adjacent-six-membered ring, and pharmaceutically acceptable salts thereof. The compounds are represented by structure Ia, below, where Zxe2x80x2 represents a 4xe2x80x2 or 5xe2x80x2 substituent on the rightmost-depicted ring, and each of Z and Zxe2x80x2 is selected from the group consisting of hydrogen, chloro, fluoro, carboxy, and methyl. 
These compounds include the 4-amino substituted compounds:
2-(benzimidazol-2xe2x80x2-yl)methyl-4-amino benzimidazole (designated herein as SNX 912);
2-(5xe2x80x2-chlorobenzimidazol-2xe2x80x2-yl)methyl-4-amino benzimidazole (designated herein as SNX 923);
2-(benzimidazol-2xe2x80x2-yl)methyl-4-amino-5-chloro benzimidazole (designated herein as SNX 947);
2-(4xe2x80x2-fluorobenzimidazol-2xe2x80x2-yl)methyl-4-amino benzimidazole (designated herein as SNX 940);
2-(5xe2x80x2-fluorobenzimidazol-2xe2x80x2-yl)methyl-4-amino benzimidazole (designated herein as SNX 942);
2-(5xe2x80x2-carboxybenzimidazol-2xe2x80x2-yl)methyl-4-amino benzimidazole (designated herein as SNX 977); and
2-(4xe2x80x2-methylbenzimidazol-2xe2x80x2-yl)methyl-4-amino benzimidazole (designated herein as SNX 944).
Compounds of formula I also forming part of the invention include the 4-nitro substituted compounds 2-(benzimidazol-2xe2x80x2-yl)methyl-4-nitro-5-chloro benzimidazole (designated herein as SNX 937) and 2-(4xe2x80x2-nitro-5xe2x80x2-chlorobenzimidazol-2xe2x80x2-yl)methyl-4-nitro-5-chloro benzimidazole (designated herein as SNX 934), and the keto-linked compounds 2-(2-indolylcarbonyl)benzimidazole (designated herein as SNX 1772) and 2,2-carbonylbisbenzimidazole (designated herein as SNX 1719).
Also within the invention are selected compounds of formula II above, represented by structure IIa: 
where each of R, Rxe2x80x2 and Rxe2x80x3 is selected from hydrogen and methyl, and W represents a two- to four-carbon alkyl chain linking the two attached nitrogen atoms to form a five- to seven-membered heterocyclic ring. Each carbon atom of the alkyl chain is unsubstituted or substituted with one or two lower alkyl groups or a hydroxyl group. Preferably, each carbon atom of the alkyl chain is unsubstituted or methyl substituted. These include the compounds:
2-(3,4,5,6-tetrahydro-5-hydroxypyrimidin-2-yl)methyl benzimidazole (designated herein as SNX-1817);
2-(3,4,5,6-tetrahydropyrimidin-2-yl)methyl benzimidazole (designated herein as SNX-1818);
2-(4,5,6,7-tetrahydro-1,3-diazepin-2-yl)methyl benzimidazole (designated herein s SNX-1819); and
1-methyl-2-[(1-methyl-4,5-dihydro-imidazol-2-yl)ethyl benzimidazole (designated herein as SNX-1771).
Also included within the invention is a compound of formula II above, 2-(1,3-dimethyl-4,5-dihydro-1H-imidazol-2-ylmethyl)-1H-benzimidazole, designated herein as SNX 980.